Modified sarcosine oxidase, process for producing the same and reagent composition using the same

ABSTRACT

A protein having been modified by addition, deletion, insertion or substitution of at least one amino acid in an amino acid sequence constituting a protein having a sarcosine oxidase activity and still having the sarcosine oxidase activity, characterized by having an improved stability in the state of a liquid compared with the unmodified one and/or having a lowered action on L-proline compared with the unmodified one. A sarcosine oxidase having at least one of the following characteristics, i.e., an action on L-proline being 0.7% or less based on sarcosine and a Km value to L-proline being 150 mM or more, when measured at 37° C. and pH 8.0; a process for producing sarcosine oxidase having an excellent substrate specificity which comprises culturing a microorganism capable of producing sarcosine oxidase and collecting the sarcosine oxidase from the culture medium; and a reagent for measuring creatinine which contains the sarcosine oxidase.

TECHNICAL FIELD

The present invention relates to sarcosine oxidase obtained by modifyinga protein having a sarcosine oxidase activity by a protein engineeringtechnique, characterized by having an improved stability in the state ofa liquid, an excellent substrate specificity and a lowered action onproline, as well as a process for producing sarcosine oxidase and areagent composition by the use thereof.

BACKGROUND ART

Sarcosine oxidase (EC 1.5.3.1) is used as an enzyme for measuringcreatine and creatinine in the body fluid, which are clinical indicatorsof diagnosis of muscular diseases and renal diseases, together with theother enzymes such as creatininase, creatinase and peroxidase. Sarcosineoxidase acts on sarcosine which is a substrate in the presence of waterand oxygen to produce glycine, formaldehyde and hydrogen peroxide.

It has been known that such sarcosine oxidase is produced by bacteriabelonging to genera Bacillus (JP-54-52789-A, JP-61-162174-A),Corynebacterium (J. Biochem., 89:599, 1981), Cylindrocarpon(JP-56-92790-A), Pseudomonas (JP-60-43379-A), and Arthrobacter(JP-2-265478-A). Technology to produce sarcosine oxidase on a largescale using a host such as Escherichia coli with a sarcosine oxidasegene obtained from these bacteria by a gene engineering technique hasbeen also reported (JP-5-115281-A, JP-6-113840-A, JP-8-238087-A).

Along with recent liquefied reagents for clinical diagnosis, variousstabilization methods of reagent components in a liquid have beeninvestigated, and also for sarcosine oxidase used for reagents formeasuring creatinine and creatine, one which is excellent in stabilityin the liquid has been desired. Our group previously reported a mutanttype of sarcosine oxidase whose stability for metal ions was improved bymodifying a wild type of sarcosine oxidase in a protein engineeringmanner (see e.g., JP-7-163341), but concerning long term storagestability in a diagnostic reagent, more improvement has beenanticipated.

Furthermore, it has been known that conventional sarcosine oxidase alsoacts on proline which is one amino acid present in blood, and it hasbeen pointed out that this can cause a true or false difference uponmeasuring creatinine and creatine (Rinsho Kagaku, 20:144-152, 1991;Seibutsu Shiryo Bunseki, 17:332-337, 1994). In order to solve thisproblem, our group reported sarcosine oxidase having a lowered action onproline by modifying the wild type of sarcosine oxidase in the proteinengineering manner (JP-10-248572-A), but the action thereof on sarcosinewhich is an original substrate has been unclear, and more improvementhas been desired.

It is an object of the present invention to provide modified sarcosineoxidase having an improved stability in a liquid.

It is another object of the present invention to provide sarcosineoxidase having a low reactivity to proline and having an excellentsubstrate specificity.

It is another object of the present invention to provide sarcosineoxidase having a lowered action on proline.

DISCLOSURE OF THE INVENTION

As a result of an extensive study for accomplishing the above objects,the present inventors have found that sarcosine oxidase can be modifiedto have an improved stability in a liquid or have a lowered action onproline without impairing an action on sarcosine.

Furthermore, the present inventors have found that sarcosine oxidase canbe modified to keep high affinity with sarcosine and have the loweredaction on L-proline, and thereby completed the present invention.

That is, the present invention is composed of the followingconstitution.

[1] Modified sarcosine oxidase which is a protein converted by adding,deleting, inserting or substituting at least one amino acid in an aminoacid sequence constituting a protein having a sarcosine oxidaseactivity, characterized by having the sarcosine oxidase activity andhaving an improved stability in a liquid compared with one beforeconversion.

[2] The modified sarcosine oxidase according to [1] characterized inthat at least one amino acid in the amino acid sequence constituting theprotein having the sarcosine oxidase activity is substituted with otheramino acid.

[3] The modified sarcosine oxidase according to [1] wherein the proteinhaving the sarcosine oxidase activity has homology of 50% or more to theamino acid sequence according to SEQ ID NO:1.

[4] The modified sarcosine oxidase according to [1] wherein the proteinhaving the sarcosine oxidase activity has homology of 80% or more to theamino acid sequence according to SEQ ID NO:1.

[5] The modified sarcosine oxidase according to [1] wherein the proteinhaving the sarcosine oxidase activity has the amino acid sequenceaccording to SEQ ID NO:1.

[6] The modified sarcosine oxidase according to [1] characterized inthat at least one amino acid in a region corresponding to positions 155to 250 in the amino acid sequence according to SEQ ID NO:1 issubstituted with other amino acid.

[7] The modified sarcosine oxidase according to [1] characterized inthat at least one amino acid in a region corresponding to positions 82to 92 or 354 to 366 in the amino acid sequence according to SEQ ID NO:1is substituted with other amino acid.

[8] The modified sarcosine oxidase according to [1] characterized inthat at least one amino acid selected from the group consisting ofregions corresponding to positions 89, 155, 166, 204, 213, 233, 240, 250and 364 in the amino acid sequence according to SEQ ID NO:1 issubstituted with other amino acid.

[9] The modified sarcosine oxidase according to [1] characterized inthat lysine at position 89 in the amino acid sequence according to SEQID NO:1 is substituted with arginine.

[10] The modified sarcosine oxidase according to [1] characterized inthat cysteine at position 155 in the amino acid sequence according toSEQ ID NO:1 is substituted with isoleucine.

[11] The modified sarcosine oxidase according to [1] characterized inthat asparagine at position 166 in the amino acid sequence according toSEQ ID NO:1 is substituted with lysine.

[12] The modified sarcosine oxidase according to [1] characterized inthat methionine at position 204 in the amino acid sequence according toSEQ ID NO:1 is substituted with alanine.

[13] The modified sarcosine oxidase according to [1] characterized inthat serine at position 213 in the amino acid sequence according to SEQID NO:1 is substituted with proline.

[14] The modified sarcosine oxidase according to [1] characterized inthat cysteine at position 233 in the amino acid sequence according toSEQ ID NO:1 is substituted with serine.

[15] The modified sarcosine oxidase according to [1] characterized inthat asparagine at position 240 in the amino acid sequence according toSEQ ID NO:1 is substituted with tyrosine.

[16] The modified sarcosine oxidase according to [1] characterized inthat glutamic acid at position 250 in the amino acid sequence accordingto SEQ ID NO:1 is substituted with glutamine.

[17] The modified sarcosine oxidase according to [1] characterized inthat alanine at position 364 in the amino acid sequence according to SEQID NO:1 is substituted with valine.

[18] Modified sarcosine oxidase which is a protein converted by adding,deleting, inserting or substituting at least one amino acid in an aminoacid sequence constituting a protein having a sarcosine oxidaseactivity, characterized by having the sarcosine oxidase activity andhaving a lowered action on L-proline compared with one beforeconversion.

[19] The modified sarcosine oxidase according to [18] characterized inthat at least one amino acid in the amino acid sequence constituting theprotein having the sarcosine oxidase activity is substituted with otheramino acid.

[20] The modified sarcosine oxidase according to [18] wherein theprotein having the sarcosine oxidase activity has homology of 50% ormore to the amino acid sequence according to SEQ ID NO:1.

[21] The modified sarcosine oxidase according to [18] wherein theprotein having the sarcosine oxidase activity has homology of 80% ormore to the amino acid sequence according to SEQ ID NO:1.

[22] The modified sarcosine oxidase according to [18] wherein theprotein having the sarcosine oxidase activity has the amino acidsequence according to SEQ ID NO:1.

[23] The modified sarcosine oxidase according to [18] characterized inthat at least one amino acid at positions 82 to 152 and 216 to 328 inthe amino acid sequence according to SEQ ID NO:1 is substituted withother amino acid.

[24] The modified sarcosine oxidase according to [18] characterized inthat at least one amino acid at positions 82 to 97 and 313 to 328 in theamino acid sequence according to SEQ ID NO:1 is substituted with otheramino acid.

[25] The modified sarcosine oxidase according to [18] characterized inthat at least one amino acid selected from the group consisting ofpositions 89, 94 and 322 in the amino acid sequence according to SEQ IDNO:1 is substituted with other amino acid.

[26] The modified sarcosine oxidase according to [18] characterized inthat lysine at position 89 in the amino acid sequence according to SEQID NO:1 is substituted with arginine.

[27] The modified sarcosine oxidase according to [18] characterized inthat valine at position 94 in the amino acid sequence according to SEQID NO:1 is substituted with glycine.

[28] The modified sarcosine oxidase according to [18] characterized inthat lysine at position 322 in the amino acid sequence according to SEQID NO:1 is substituted with arginine.

[29] The modified sarcosine oxidase according to [18] characterized inthat a Km value for sarcosine after the modification is within 3 timescompared with the unmodified one.

[30] The modified sarcosine oxidase according to [18] characterized inthat a Km value for sarcosine after the modification is within 1.5 timescompared with the unmodified one.

[31] Sarcosine oxidase characterized by having at least one of thefollowing characteristics under a measurement condition at 37° C. and pH8.0:

-   -   action on L-proline: 0.7% or less based on sarcosine; and    -   Km value for L-proline: 150 mM or more.

[32] The sarcosine oxidase according to [31] characterized by having atleast one of the following characteristics under a measurement conditionat 37° C. and pH 8.0:

-   -   action on L-proline: 0.5% or less based on sarcosine; and    -   Km value for L-proline: 200 mM or more.

[33] The sarcosine oxidase according to [31] wherein the Km value forsarcosine is 10 mM or less.

[34] The sarcosine oxidase according to [31] wherein the Km value forsarcosine is 5 mM or less.

[35] A gene encoding the modified sarcosine oxidase according to any oneof [1] to [17] and [18] to [30].

[36] A vector containing the gene according to [35].

[37] A transformant transformed with the vector according to [36].

[38] A process for producing modified sarcosine oxidase characterized inthat the transformant according to [37] is cultured and the sarcosineoxidase is collected from the culture.

[39] A process for producing modified sarcosine oxidase which isexcellent in substrate specificity, characterized in that amicroorganism having a production capacity of the sarcosine oxidaseaccording to any one of [31] to [34] is cultured and the sarcosineoxidase is collected from the culture.

[40] A reagent for measuring creatine containing the sarcosine oxidaseaccording to any one of [1] to [17], [18] to [30] and [31] to [34].

[41] A reagent for measuring creatinine containing the sarcosine oxidaseaccording to any one of [1] to [17], [18] to [30] and [31] to [34].

The present invention will be described in detail below.

The modified sarcosine oxidase of the present invention is useful foranalysis of creatine and creatinine in the clinical examination field.

One embodiment of the present invention is a protein modified byaddition, deletion, insertion or substitution of at least one amino acidin an amino acid sequence constituting a protein having a sarcosineoxidase activity, and modified sarcosine oxidase characterized by havingthe sarcosine oxidase activity, an improved stability in a liquidcompared with an unmodified one, a sufficiently lowered action onproline compared with sarcosine which is an original substrate, orhaving the sarcosine oxidase activity and a lowered action on prolinecompared with the unmodified one.

The action on proline can be obtained by a relative ratio (%) of anenzymatic activity using L-proline as the substrate to an enzymaticactivity using sarcosine as the substrate. In the sarcosine oxidase ofthe present invention, the action on L-proline is 0.7% or less andpreferably 0.5% or less based on the action on sarcosine.

In another embodiment of the present invention, the sarcosine oxidasehas a high Km value (Michaelis-Menten constant) for proline, and isunlikely to be affected by proline in a sample when applied to a reagentfor measuring creatinine and creatine. The sarcosine oxidase of thepresent invention has the Km value of 150 mM or more and preferably 200mM or more for L-proline.

In another embodiment of the present invention, the sarcosine oxidase ofthe present invention has the Km value of preferably 10 mM or less andmore preferably 5 mM or less for sarcosine in terms of suppressing anecessary amount to be added in the measurement reagent and takingadvantage of the high substrate specificity.

The sarcosine oxidase of the present invention is not particularlylimited as long as it has the above characters. For example, enzymesderived from microorganisms and mammals can be used. Enzymes obtained bymodifying the publicly known sarcosine oxidase using geneengineering/protein engineering technology and enzymes whose property isimproved by chemical modification are also included.

The modified sarcosine oxidase in one embodiment of the presentinvention is characterized in that the stability in the liquid isfurther improved compared with the unmodified one. The stability in theliquid in the present invention means, for example, a proportion of aresidual enzyme activity after the modified enzyme has been dissolved inan appropriate buffer and stored at an appropriate temperature for acertain time period.

The “appropriate buffer” is not particularly limited as long as a typethereof and the temperature is selected so that sufficient buffercapacity is kept at pH of around 7 to 8 which is an optimal pH of thesarcosine oxidase. Preferably, 50 mM potassium phosphate buffer (pH 7.5)or 50 mM PIPES-NaOH buffer (pH 7.5) is selected. Furthermore,surfactants, salts, chelating agents and preservatives may be containedin the buffer if necessary.

A condition of “storage at the appropriate temperature for the certaintime period” is not particularly limited, but preferably, a condition ofan acceleration (harshness) test is selected with long term storagestability in a liquid diagnostic reagent in mind. Specifically, “storageat 40° C. for 3 days” or “storage at 60° C. for 30 min” is included.When time permits, the storage under a cooling condition at 2 to 10° C.commonly used as the temperature at which the liquid diagnostic drug isactually stored for a long time, for 6 months or more may be selected.

A concentration of the sarcosine oxidase in the storage is notparticularly limited, and the concentration of 1 to 30 U/mL ispreferably selected on the assumption of the concentration typicallyused for the diagnostic reagent. More preferably, the concentration of 5to 20 U/mL is selected.

The “stability being further improved compared with the unmodified one”refers to that an activity keeping proportion after the storage for thecertain period is higher than an activity keeping proportion of anunmodified enzyme when measured under the same condition.

One embodiment of the present invention is modified sarcosine oxidasewhere a residual enzyme activity proportion after storing in 50 mMpotassium phosphate buffer (pH 7.5) at 60° C. for 30 min is improvedcompared with the unmodified one. Another embodiment is the modifiedsarcosine oxidase where the residual enzyme activity proportion afterstoring in 50 mM PIPES-NaOH buffer (pH 7.5) containing 2 mM EDTA, 50 mMNaCl, 0.1% (w/v) 2-methylisothiazolone, and 0.1% (w/v) TritonX-100 at40° C. for 3 days is improved compared with the unmodified one.

The modified sarcosine oxidase in one embodiment of the presentinvention is characterized in that a reactivity to proline is loweredcompared with the unmodified one. The reactivity to proline means therelative ratio of the enzyme activity using proline as the substrate tothe enzyme activity using sarcosine which is the original substrate asthe substrate. And, as long as the reactivity to proline is lowered,even if a specific activity using sarcosine as the substrate is changed,the enzyme is included in the modified sarcosine oxidase of the presentinvention. In the modified sarcosine oxidase of the present invention,the Km value for sarcosine may be changed, but when applied to thereagent for measuring creatinine and creatine, it causes loweredreactivity, and thus, the Km value for sarcosine is preferably within 3times, and more preferably within 1.5 times of that before themodification.

Sarcosine oxidase used for the modification of the present invention isnot particularly limited, and for example, the sarcosine oxidase derivedfrom bacteria belonging to genera Bacillus, Pseudomonas andCorynebacterium known publicly can be used.

An example in which sarcosine oxidase (JP-2-265478-A) derived fromArthrobacter sp. TE1826 (Accession No. 10637, Fermentation ResearchInstitute, the Agency of Industrial Science and Technology) was modifiedin a protein engineering manner is shown as one example in the presentinvention.

The group of the present inventors have successfully isolated asarcosine oxidase gene from chromosomal DNA extracted from Arthrobactersp. TE1826, determined an entire DNA structure thereof (described inJournal of Fermentation and Bioengineering, Vol. 75, No. 4:239-244,1999), successfully produced the sarcosine oxidase in transformants at ahigh density by a gene engineering technique, and enabled toinexpensively supply the sarcosine oxidase with high purity on a largescale (JP-6-113840-A). An amino acid sequence of the sarcosine oxidasefrom Arthrobacter sp. TE1826 is shown in SEQ ID NO:1. A DNA sequenceencoding this amino acid sequence is shown in SEQ ID NO:2.

But, the present invention is not limited to the modified sarcosineoxidase having the amino acid sequence described in SEQ ID NO:1, and maybe the other modified protein having the sarcosine oxidase activity.Suitable examples of the other protein having the sarcosine oxidaseactivity include sarcosine oxidase whose three dimensional structure issimilar to that of the sarcosine oxidase having the amino acid sequencedescribed in SEQ ID NO:1, and specifically include other proteins having50% or more homologous amino acid sequence, more preferably 80% or morehomologous amino acid sequence, and having the sarcosine oxidaseactivity. This is based on the respect that enzyme proteins having 50 to80% or more homology in the amino acid sequence and exhibiting the samecatalytic activity are often similar in the three dimensional structureand often have the same amino acid residues involved in the substratespecificity and the same reaction mechanism.

In the modified sarcosine oxidase, as long as the enzyme activity and/orstability which are essence of the enzyme property of the presentinvention are not impaired, one or more amino acids may be furtherdeleted, substituted or added. Specifically, one which has added ahistidine tag at the N- or C-terminus of the amino acid sequence inorder to simplify the purification of sarcosine oxidase is exemplified(e.g., “Jikken Igaku” 20:479-482, 2002).

The homology in the amino acid sequences in the present invention can becalculated using publicly known gene analysis software (e.g.,Genetyx-win ver. 3, Genetyx Corporation). The homology refers to apercentage of identical amino acid residues in the range havingsimilarity to the amino acid sequence to be compared.

Another embodiment of the present invention is the modified sarcosineoxidase characterized in that at least one amino acid in the amino acidsequence described in SEQ ID NO:1 is substituted with other amino acid,and having the improved stability in the liquid compared with theunmodified one.

Another embodiment of the present invention is the modified sarcosineoxidase characterized in that at least one amino acid in sitescorresponding to positions 155 to 250 in the amino acid sequence fromArthrobacter sp. TE1826 described in SEQ ID NO:1 or sites correspondingto positions 155 to 250 in the amino acid sequence described in SEQ IDNO:1 in sarcosine oxidase from other than Arthrobacter sp. TE1826 issubstituted with the other amino acid, and having the improved stabilityin the liquid compared with the unmodified one.

Another embodiment of the present invention is the modified sarcosineoxidase characterized in that at least one amino acid in sitescorresponding to positions 82 to 92 in the amino acid sequence fromArthrobacter sp. TE1826 described in SEQ ID NO:1 or sites correspondingto positions 82 to 92 of the amino acid sequence described in SEQ IDNO:1 in sarcosine oxidase from other than Arthrobacter sp. TE1826 issubstituted with the other amino acid, and having the improved stabilityin the liquid compared with the unmodified one.

There is a report of the sarcosine oxidase whose three dimensionalstructure has been demonstrated by X-ray crystal analysis (e.g.,“Structure” Vol. 7, No. 3:331-345, 1999). In accordance with the report,the sarcosine oxidase has the homology to the amino acid sequencedescribed in SEQ ID NO:1, and it has been speculated that the sitescorresponding to positions 82 to 92 in the amino acid sequence describedin SEQ ID NO:1 which is the amino acid sequence of the sarcosine oxidasefrom Arthrobacter sp. TE1826 or the sites corresponding to positions 82to 92 in the amino acid sequence described in SEQ ID NO:1 in sarcosineoxidase from other than Arthrobacter sp. TE1826 constitute a linked siteof a catalytic domain and an FAD binding domain of the sarcosineoxidase.

Another embodiment of the present invention is the modified sarcosineoxidase characterized in that at least one amino acid in sitescorresponding to positions 354 to 366 presumed to constitute α-helixcontaining the position 364 in the amino acid sequence from Arthrobactersp. TE1826 described in SEQ ID NO:1 or sites corresponding to positions354 to 366 in the amino acid sequence described in SEQ ID NO:1 in thesarcosine oxidase from other than Arthrobacter sp. TE1826 is substitutedwith the other amino acid, and having the improved stability in theliquid compared with the unmodified one.

Preferable is the modified sarcosine oxidase where at least one aminoacid selected from the group consisting of positions 89, 155, 166, 204,213, 233, 240, 250 and 364 in the amino acid sequence described in SEQID NO:1 or corresponding positions in the other sarcosine oxidase issubstituted with the other amino acid.

More preferable is the modified sarcosine oxidase where at least oneamino acid selected from the following group is substituted with theother amino acid. The modified sarcosine oxidase where lysine atposition 89 is substituted with arginine, cysteine at position 155 issubstituted with isoleucine, asparagine at position 166 is substitutedwith lysine, methionine at position 204 is substituted with alanine,serine at position 213 is substituted with proline, cysteine at position233 is substituted with serine, asparagine at position 240 issubstituted with tyrosine, glutamic acid at position 250 is substitutedwith glutamine or alanine at position 364 is substituted with valine isexemplified.

Another embodiment of the present invention is the modified sarcosineoxidase where the protein having the sarcosine oxidase activity has theamino acid sequence described in SEQ ID NO:1.

Another embodiment of the present invention is the modified sarcosineoxidase where at least one amino acid in a region which constitutes thecatalytic domain and the linked site of the catalytic domain and the FADbinding domain is substituted with the other amino acid. It has beenpredicted that positions 82 to 152 and positions 216 to 328 in the aminoacid sequence described in SEQ ID NO:1 constitute the catalytic domainand the linked site of the catalytic domain and the FAD binding domainof the sarcosine oxidase, from the sarcosine oxidase having the homologyto the amino acid sequence described in SEQ ID NO:1, whose threedimensional structure has been demonstrated by the X-ray crystalanalysis (e.g., “Structure” Vol. 7, No. 3:331-345, 1999).

Preferable is the modified sarcosine oxidase where at least one aminoacid in the region which constitutes the linked site of the catalyticdomain and the FAD binding domain, and a β-sheet structure of thecatalytic domain proximal thereto is substituted with the other aminoacid. It has been predicted that positions 82 to 97 and positions 313 to328 in the amino acid sequence described in SEQ ID NO:1 constitute thelinked site of the catalytic domain and the FAD binding domain, and theβ-sheet structure of the catalytic domain proximal thereto of thesarcosine oxidase (e.g., “Structure” Vol. 7, No. 3:331-345, 1999).

Another embodiment of the present invention is the modified sarcosineoxidase where at least one amino acid selected from the group consistingof positions 89, 94 and 322 in the amino acid sequence described in SEQID NO:1 is substituted with the other amino acid.

Among them, preferable is the modified sarcosine oxidase where lysine atposition 89 is substituted with arginine, valine at position 94 issubstituted with glycine or lysine at position 322 is substituted witharginine in the amino acid sequence described in SEQ ID NO:1.

Another embodiment of the present invention is a gene encoding the abovemodified sarcosine oxidase, a vector containing the gene, a transformanttransformed with the vector, and further a process for producing themodified sarcosine oxidase characterized in that the transformant iscultured and the sarcosine oxidase is collected from the culture.

The process for producing the sarcosine oxidase of the present inventionis not particularly limited, and when the publicly known enzyme isimproved using the protein engineering technique, it is possible toproduce by the procedure shown below. As a method for modifying theamino acid sequence which constitutes the protein having the sarcosineoxidase activity, the typically performed technique for modifying geneinformation is used. That is, a DNA having the gene information of themodified protein is made by converting a certain base of a DNA havingthe gene information of the protein, or inserting or deleting a certainbase. Specific methods for converting the base in the DNA moleculeinclude the use of commercially available kits (Transformer Mutagenesiskit supplied from Clonetech; EXOIII/Mung Bean Deletion Kit supplied fromStratagene; QuickChange Site Directed Mutagenesis Kit supplied fromStratagene) or the utilization of polymerase chain reaction (PCR).

The DNA having the gene information of the produced modified protein isligated to a plasmid and transfected into a host microorganism, whichthen becomes a transformant which produces the modified protein. WhenEscherichia coli is used as the host microorganism, pBluescript andpUC18 can be used as the plasmid. As the host microorganism, forexample, Escherichia coli W3110, Escherichia coli C600, Escherichia coliJM109, Escherichia coli DH5α and the like can be utilized. As the methodfor transfecting a recombinant vector into the host microorganism, whenthe host is the microorganism belonging to genus Escherichia, it ispossible to employ the method in which the recombinant DNA istransfected in the presence of calcium ions, and further, anelectroporation method may be used. Furthermore, commercially availablecompetent cells (e.g., Competent High JM109 supplied from Toyobo Co.,Ltd.) may be used.

The modified protein can be stably produced on a large scale byculturing the microorganism which is the transformant obtained in thisway in a nutrient medium. For a culture form of the microorganism whichis the transformant, a culture condition could be selected inconsideration of nutritional physiological natures of the host,typically the liquid culture is often performed, but industrially, it isadvantageous to perform an aeration stirring culture. As nutritioussources of the medium, those used for the culture of the microorganismcan be widely used. Carbon sources may be carbon compounds capable ofbeing utilized, and for example, glucose, sucrose, lactose, maltose,fructose, treacle, pyruvic acid and the like are used. Nitrogen sourcesmay be usable nitrogen compounds, and for example, peptone, meatextract, yeast extract, hydrolyzed casein, alkali extract of soy beancake, and the like are used. Additionally, phosphate salts, carbonatesalts, sulfate salts, salts of magnesium, calcium, potassium, iron,manganese and zinc, certain amino acids, certain vitamins are used ifnecessary. A culture temperature can be appropriately changed in therange where the bacteria grow and produce the modified protein, and inthe case of Escherichia coli it is preferably about 20 to 42° C. Aculture period is changed more or less depending on the condition, andis typically about 6 to 48 hours because the culture may beappropriately terminated when the modified protein has been yieldmaximally. Medium pH can be appropriately changed in the range where thebacteria grow and produce the modified protein, and in particular ispreferably about pH 6.0 to 9.0.

The sarcosine oxidase can be collected by culturing the microorganismhaving the sarcosine oxidase of the present invention as a wild typeenzyme under the culture condition suitable for growth of eachmicroorganism using an appropriate nutritious medium. At that time, inorder to induce the enzyme expression, it is desirable to add sarcosine,creatine and dimethyl glycine at an appropriate amount into the medium.

A culture fluid containing microbial cells which produce the modifiedprotein in the culture can be employed as it is and utilized, but ingeneral, when the modified protein is present in the culture fluid inaccordance with standard methods, a modified protein-containing solutionis utilized after separating from the microbial cells by filtration,centrifugation and the like. When the modified protein is present in themicrobial cells, the microbial cells are collected by means offiltration, centrifugation and the like from the culture, then thesemicrobial cells are disrupted by a mechanical method or an enzymaticmethod such as lysosome, and if necessary the modified protein issolubilized by adding a chelating agent such as EDTA or a surfactant toseparate/collect as an aqueous solution.

The modified protein-containing solution obtained in this way may beprecipitated by, for example, concentration under reduced pressure,concentration via a membrane, salting out by ammonium sulfate or sodiumsulfate, or fractional precipitation by hydrophilic organic solvent suchas methanol, ethanol and acetone. Treatment with heat and treatmenttaking advantage of isoelectric point are also effective purificationmeans. The purified modified protein can be obtained by gel filtration,adsorption chromatography, ion exchange chromatography or affinitychromatography by an adsorption agent and a gel filtrating agent.

Another embodiment of the present invention is a reagent for measuringcreatine or creatinine containing the above modified sarcosine oxidase.In the reagent for measuring creatine or creatinine, an active period ofthe reagent can be prolonged or measurement accuracy can be enhanced byusing the modified sarcosine oxidase having the improved liquidstability and the lowered action on proline. An effect degree of prolinecan be suppressed to less than 7% and preferably less than 5%.

The reagent for measuring creatine of the present invention includes themodified sarcosine oxidase having the improved stability in the liquid,the small substrate specificity for proline or the lowered reaction toproline, creatine amidinohydrolase, peroxidase and a reagent fordetecting hydrogen peroxide.

The reagent for measuring creatinine includes the modified sarcosineoxidase having the improved stability in the liquid, the small substratespecificity for proline or the lowered reaction to proline, creatinineamidohydrolase, creatine amidinohydrolase, peroxidase and a reagent fordetecting hydrogen peroxide.

The reagent for detecting hydrogen peroxide is a reagent which measureshydrogen peroxide produced by the sarcosine oxidase as a producedpigment in the presence of peroxidase, and includes an oxidativecoloring reagent and if necessary a coupler such as 4-aminoantipyrineand 3-methyl-2-benzothiazolinone.

The reagent for detecting hydrogen peroxide of the present invention isnot particularly limited, and various commercially available ones can beused. Furthermore, in the above reagent for measuring creatine orcreatinine, metal salts, proteins, amino acids, sugars, organic acidsand the like can be also used as stabilizing agents. Preservatives andsurfactants are usually added in the range where no harmful effect isgiven to reagent performance, and used together with an appropriatebuffer. One or more are selected for types, concentrations and pH of thebuffer depending on the purposes such as storage of each reagentingredient and enzyme reaction, and when using any buffer, pH at theenzyme reaction is preferably in the range of 5.0 to 10.0.

In the present invention, the sarcosine oxidase activity is measuredunder the following condition.

<Reagents>

-   -   100 mM Tris-HCl buffer (pH 8.0) (containing 200 mM sarcosine and        0.1% Triton X-100)    -   0.1% 4-aminoantipyrine    -   0.1% phenol    -   25 U/mL peroxidase        <Measurement Condition>

A reaction mixture is prepared by mixing the above Tris-HCl buffer,4-aminoantipyrine solution, phenol solution and peroxidase solution at aratio of 5:1:2:2. The reaction mixture (1 mL) is taken to a test tube,preliminarily warmed at 37° C. for about 5 min, and 0.05 mL of an enzymesolution is added to start the reaction. The reaction at 37° C. isperformed accurately for 10 min, then 2.0 mL of an aqueous solution of0.25% SDS is added to stop the reaction, and an absorbance at 500 nm ofthis solution is measured. In a blind test, distilled water instead ofthe enzyme solution is added to the reagent mixture, and the samemanipulation is followed to measure the absorbance. Under the abovecondition, an amount of the enzyme which produces 1 μmol of hydrogenperoxide per min is made one unit. The reactivity to proline wasmeasured as a relative ratio of activities when sarcosine in the abovereagent was replaced by L-proline at the same concentration.

In accordance with the present invention, it became possible to supplythe modified sarcosine oxidase having the improved liquid stability, themodified sarcosine oxidase having the lowered action on proline, and themodified sarcosine oxidase having the small action on L-proline and theexcellent substrate specificity by modifying the protein having thesarcosine oxidase activity by the protein engineering technique. By theuse of the modified sarcosine oxidase of the present invention as theenzyme for measuring creatine and creatinine in the body fluid, whichare clinical indicators of the diagnosis for muscular diseases and renaldiseases, it becomes possible to accurately measure creatine andcreatinine without being affected by coexisting substance (e.g.,proline), and the liquid stability of the reagent can be improved.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be specifically described with reference tothe following Examples, but the invention is not limited thereto.

For example, among 13 types of modified sarcosine oxidases shown inExample 3A and 9 types of modified sarcosine oxidases shown in Example3B described later, SAOM1 is a mutant where lysine at position 89 issubstituted with arginine in the amino acid sequence of SEQ ID NO:1 andSAOM2 is a mutant where valine at position 94 is substituted withglycine in the amino acid sequence of SEQ ID NO:1. However, one orseveral amino acids may further be deleted, substituted or added withinthe range where the performance of the mutant is not substantiallyaffected. This is the same for mutants other than SAOM1 and SAOM2.

EXAMPLE 1 Construction of Expression Plasmid for Sarcosine Oxidase

An expression plasmid, pSAOEP3 for sarcosine oxidase derived fromArthrobacter sp. TE1826 strain was constructed in accordance with themethod described in JP-7-163341-A. This expression plasmid contains aninserted DNA fragment of about 1.7 Kbp containing a gene encoding thesarcosine oxidase of TE1826 at a multiple cloning site of pUC18. Anucleotide sequence thereof is shown in SEQ ID NO:2, and an amino acidsequence of the sarcosine oxidase deduced from the nucleotide sequenceis shown in SEQ ID NO:1.

EXAMPLE 2A Preparation of Modified Sarcosine Oxidase Gene

A recombinant plasmid (pSAOM1A) encoding modified sarcosine oxidasewhere lysine at position 89 was substituted with arginine in the aminoacid sequence of SEQ ID NO:1 was obtained by using the expressionplasmid pSAOEP3 containing a sarcosine oxidase gene and a syntheticoligonucleotide in SEQ ID NO:3 and a synthetic oligonucleotidecomplementary thereto, using QuickChange™ Site-Directed Mutagenesis Kit(supplied from Stratagene), manipulating in accordance with theprotocol, and further sequencing.

A recombinant plasmid (pSAOM2A) encoding modified sarcosine oxidasewhere cysteine at position 155 was substituted with isoleucine in theamino acid sequence of SEQ ID NO:1 was obtained by using pSAOEP3 and asynthetic oligonucleotide in SEQ ID NO:4 and a synthetic oligonucleotidecomplementary thereto, using QuickChange™ Site-Directed Mutagenesis Kit(supplied from Stratagene), and manipulating in the same way as in theabove.

A recombinant plasmid (pSAOM3A) encoding modified sarcosine oxidasewhere asparagine at position 166 was substituted with lysine in theamino acid sequence of SEQ ID NO:1 was obtained by using pSAOEP3 and asynthetic oligonucleotide in SEQ ID NO:5 and a synthetic oligonucleotidecomplementary thereto, and manipulating in the same way as in the above.

A recombinant plasmid (pSAOM4A) encoding modified sarcosine oxidasewhere methionine at position 204 was substituted with alanine in theamino acid sequence of SEQ ID NO:1 was obtained by using pSAOEP3 and asynthetic oligonucleotide in SEQ ID NO:6 and a synthetic oligonucleotidecomplementary thereto, and manipulating in the same way as in the above.

A recombinant plasmid (pSAOM5A) encoding modified sarcosine oxidasewhere serine at position 213 was substituted with proline in the aminoacid sequence of SEQ ID NO:1 was obtained by using pSAOEP3 and asynthetic oligonucleotide in SEQ ID NO:7 and a synthetic oligonucleotidecomplementary thereto, and manipulating in the same way as in the above.

A recombinant plasmid (pSAOM6A) encoding modified sarcosine oxidasewhere cysteine at position 233 was substituted with serine in the aminoacid sequence of SEQ ID NO:1 was obtained by using pSAOEP3 and asynthetic oligonucleotide in SEQ ID NO:8 and a synthetic oligonucleotidecomplementary thereto, and manipulating in the same way as in the above.

A recombinant plasmid (pSAOM7A) encoding modified sarcosine oxidasewhere asparagine at position 240 was substituted with tyrosine in theamino acid sequence of SEQ ID NO:1 was obtained by using pSAOEP3 and asynthetic oligonucleotide in SEQ ID NO:9 and a synthetic oligonucleotidecomplementary thereto, and manipulating in the same way as in the above.

A recombinant plasmid (pSAOM8A) encoding modified sarcosine oxidasewhere glutamic acid at position 250 was substituted with glutamine inthe amino acid sequence of SEQ ID NO:1 was obtained by using pSAOEP3 anda synthetic oligonucleotide in SEQ ID NO:10 and a syntheticoligonucleotide complementary thereto, and manipulating in the same wayas in the above.

A recombinant plasmid (pSAOM9A) encoding modified sarcosine oxidasewhere alanine at position 364 was substituted with valine in the aminoacid sequence of SEQ ID NO:1 was obtained by using pSAOEP3 and asynthetic oligonucleotide in SEQ ID NO:11 and a syntheticoligonucleotide complementary thereto, and manipulating in the same wayas in the above.

A recombinant plasmid (pSAOM10A) encoding modified sarcosine oxidasewhere lysine at position 89 was substituted with arginine and serine atposition 213 was substituted with proline in the amino acid sequence ofSEQ ID NO:1 was obtained by using pSAOM1A and a syntheticoligonucleotide in SEQ ID NO:7 and a synthetic oligonucleotidecomplementary thereto, and manipulating in the same way as in the above.

A recombinant plasmid (pSAOM11A) encoding modified sarcosine oxidasewhere lysine at position 89 was substituted with arginine, serine atposition 213 was substituted with proline and glutamic acid at position250 was substituted with glutamine in the amino acid sequence of SEQ IDNO:1 was obtained by using pSAOM10A and a synthetic oligonucleotide inSEQ ID NO:10 and a synthetic oligonucleotide complementary thereto, andmanipulating in the same way as in the above.

A recombinant plasmid (pSAOM12A) encoding modified sarcosine oxidasewhere lysine at position 89 was substituted with arginine, cysteine atposition 155 was substituted with isoleucine, asparagine at position 166was substituted with lysine, serine at position 213 was substituted withproline, glutamic acid at position 250 was substituted with glutamineand alanine at position 364 was substituted with valine in the aminoacid sequence of SEQ ID NO:1 was obtained by using pSAOM11A andsynthetic oligonucleotides in SEQ ID NOS:4, 5, 11 and syntheticoligonucleotides complementary thereto, and manipulating in the same wayas in the above.

A recombinant plasmid (pSAOM13A) encoding modified sarcosine oxidasewhere lysine at position 89 was substituted with arginine, methionine atposition 204 was substituted with alanine, serine at position 213 wassubstituted with proline, cysteine at position 233 was substituted withserine, asparagine at position 240 was substituted with tyrosine, andglutamic acid at position 250 was substituted with glutamine in theamino acid sequence of SEQ ID NO:1 was obtained by using pSAOM11A andsynthetic oligonucleotides in SEQ ID NOS:6, 8, 9 and syntheticoligonucleotides complementary thereto, and manipulating in the same wayas in the above.

EXAMPLE 3A Preparation of Modified Sarcosine Oxidases

Competent cells of Escherichia coli JM109 were transformed with eachrecombinant plasmid of pSAOM1A, pSAOM2A, pSAOM3A, pSAOM4A, pSAOM5A,pSAOM6A, pSAOM7A, pSAOM8A, pSAOM9A, pSAOM10A, pSAOM11A, pSAOM12A, andpSAOM13A to obtain the transformants.

Terrific broth (400 mL) was dispensed in a 2 L Sakaguchi flask,autoclaved at 121° C. for 20 min, cooled, and subsequently ampicillinseparately sterilized and filtrated was added at 100 μg/mL. A culturefluid (5 mL) of Escherichia coli JM109 (pSAOM1) previously cultured inLB medium containing 100 μg/mL of ampicillin at 30° C. for 16 hours wasinoculated to this medium, which was then cultured with aeration andstirring at 30° C. for 20 hours. At the completion of the culture, asarcosine oxidase activity was about 9.5 U/mL per 1 mL of the culturefluid in the above activity measurement.

The above microbial cells were collected by centrifugation, suspended in20 mM phosphate buffer (pH 7.5), subsequently disrupted ultrasonically,and further centrifuged to yield a supernatant as a crud enzymesolution. Nucleic acids were removed using polyethyleneimine from theresulting crude enzyme solution, ammonium sulfate fractionation wasgiven thereto, and then separation and purification was performed bydialyzing with 20 mM phosphate buffer (pH 7.5), applying on DEAESepharose CL-6B (supplied from Amersham Bioscience) and further treatingwith heat for one hour to yield a purified enzyme preparation. Thepreparation obtained by the present method exhibited a nearly singleband on SDS-PAGE. This mutant was designated as SAOM1A.

For transformants of Escherichia coli JM109 transformed with eachrecombinant plasmid of pSAOM2A, pSAOM3A, pSAOM4A, pSAOM5A, pSAOM6A,pSAOM7A, pSAOM8A, pSAOM9A, pSAOM10A, pSAOM11A, pSAOM12A, and pSAOM13A, apurified enzyme preparation was obtained by the same way in the above,and the obtained enzyme preparation was each designated as SAOM2A,SAOM3A, SAOM4A, SAOM5A, SAOM6A, SAOM7A, SAOM8A, SAOM9A, SAOM10A,SAOM11A, SAOM12A, and SAOM13A.

COMPARATIVE EXAMPLE 1 Preparation of Wild Type Sarcosine Oxidase

As Comparative Example, Escherichia coli JM109 was transformed withpSAOEP3, and for the resulting transformant, a purified preparation ofthe unmodified enzyme was obtained in the same way as the above.

EXAMPLE 4A Evaluation of Modified Sarcosine Oxidases 1

Mutant sarcosine oxidases (SAOM1A, SAOM2A, SAOM3A, SAOM4A, SAOM5A,SAOM6A, SAOM7A, SAOM8A) obtained in Example 3A and the sarcosine oxidaseobtained in Comparative Example 1 were each added in 50 mM potassiumphosphate buffer (pH 7.5) at 5 U/mL, and a proportion (%) of theresidual enzyme activity after storing at 60° C. for 30 min wasmeasured. The results are shown in Table 1. As is shown in Table 1, ithas been confirmed that the modified sarcosine oxidase of the presentinvention is improved in liquid stability compared with the unmodifiedone. TABLE 1 Residual Modified activity one Mutant ratio (%) SAOM1A K89R34 SAOM2A C155I 46 SAOM3A N166K 37 SAOM4A M204A 51 SAOM5A S213P 47SAOM7A N240Y 52 SAOM8A E250Q 31 Unmodified — 19

EXAMPLE 4A Evaluation of Modified Sarcosine Oxidases 2

Mutant sarcosine oxidases (SAOM1A, SAOM2A, SAOM5A, SAOM6A, SAOM7A,SAOM8A SAOM9A) obtained in Example 3A and the sarcosine oxidase obtainedin Comparative Example 1 were each added in IPES-NaOH buffer (pH 7.5)containing 2 mM dihydrogen disodium ethylenediamine tetraacetate, 50 mMNaCl, 0.1% (w/v) 2-methylisothiazolone (supplied from Roche Diagnostics)and 0.1% (w/v) Triton X-100 at 5 U/mL, and a proportion (%) of theresidual enzyme activity after storing at 40° C. for 3 days wasmeasured. The results are shown in Table 2. As is shown in Table 2, ithas been confirmed that the modified sarcosine oxidase of the presentinvention is improved in liquid stability compared with the unmodifiedone. TABLE 2 Residual Modified activity one Mutant ratio (%) SAOM1A K89R41 SAOM2A C155I 47 SAOM5A S213P 49 SAOM6A C233S 72 SAOM7A N240Y 73SAOM8A E250Q 40 SAOM9A A364V 45 Unmodified — 30

EXAMPLE 5A Evaluation of Modified Sarcosine Oxidases 3

The stability of mutant sarcosine oxidases (SAOM1A, SAOM10A, SAOM11A,SAOM12A, SAOM13A) obtained in Example 3A and the sarcosine oxidaseobtained in Comparative Example 1 in a reagent for measuring creatininewas analyzed. To 50 mM PIPES-NaOH buffer (pH 7.5) containing 1 mMdihydrogen disodium ethylenediamine tetraacetate, 50 mM sodium chloride,0.1% (w/v) 2-methylisothiazolone (supplied from Roche Diagnostics), 0.1%(w/v) Triton X-100, 0.02% (w/v) 4-aminoantipyrine, 0.02% (w/v) TOOS(supplied from Dojindo Corporate), 100 U/mL creatinine amidohydrolase(CNH-211, supplied from Toyobo Co., Ltd.), 50 U/mL creatineamidinohydrolase (CRH-221, supplied from Toyobo Co., Ltd.), and 10 U/mLperoxidase (PEO-301, supplied from Toyobo Co., Ltd.), the abovesarcosine oxidase was added at 10 U/mL, and stored at 35° C. for 2weeks, and then a proportion of the residual sarcosine oxidase activitywas measured. The results are shown in Table 3. As is shown in Table 3,it has been confirmed that the modified sarcosine oxidase of the presentinvention is improved in liquid stability in the reagent for measuringcreatinine compared with the unmodified one. TABLE 3 Residual Modifiedactivity one Mutant ratio (%) SAOM1A K89R 28 SAOM10A K89R, S213P 44SAOM11A K89R, S213P, E250Q 51 SAOM12A K89R, C155I, 77 N166K, S213P,E250Q, A364V SAOM13A K89R, M204A, 79 S213P, C233S, N240Y, E250QUnmodified — 16

EXAMPLE 6A Evaluation of Modified Sarcosine Oxidases 4

The stability of mutant sarcosine oxidases (SAOM1A, SAOM10A, SAOM11A,SAOM12A, SAOM13A) obtained in Example 3A and the sarcosine oxidaseobtained in Comparative Example 1 in a reagent for measuring creatinewas analyzed. To 50 mM PIPES-NaOH buffer (pH 7.5) containing 1 mMdihydrogen disodium ethylenediamine tetraacetate, 50 mM sodium chloride,0.1% (w/v) 2-methylisothiazolone (supplied from Roche Diagnostics), 0.1%(w/v) Triton X-100, 0.02% (w/v) 4-aminoantipyrine, 0.02% (w/v) TOOS(supplied from Dojindo Corporate), 50 U/mL creatine amidinohydrolase(CRH-221, supplied from Toyobo Co., Ltd.), and 10 U/mL peroxidase(PEO-301, supplied from Toyobo Co., Ltd.), the above sarcosine oxidasewas added at 10 U/mL, and stored at 35° C. for 2 weeks, and then aproportion of the residual sarcosine oxidase activity was measured. Theresults are shown in Table 3. As is shown in Table 3, it has beenconfirmed that the modified sarcosine oxidase of the present inventionis improved in liquid stability in the reagent for measuring creatinecompared with the unmodified one. TABLE 4 Residual Modified activity oneMutant ratio (%) SAOM1A K89R 31 SAOM10A K89R, S213P 44 SAOM11A K89R,S213P, E250Q 52 SAOM12A K89R, C155I, 80 N166K, S213P, E250Q, A364VSAOM13A K89R, M204A, 77 S213P, C233S, N240Y, E250Q Unmodified — 14(control)

EXAMPLE 2B Preparation of Modified Sarcosine Oxidase Gene

A recombinant plasmid (pSAOM1B) encoding modified sarcosine oxidasewhere lysine at position 89 was substituted with arginine in the aminoacid sequence of SEQ ID NO:1 was obtained by using the expressionplasmid pSAOEP3 containing a sarcosine oxidase gene and a syntheticoligonucleotide in SEQ ID NO:3 and a synthetic oligonucleotidecomplementary thereto, using QuickChange™ Site-Directed Mutagenesis Kit(supplied from Stratagene), manipulating in accordance with theprotocol, and further sequencing.

A recombinant plasmid (pSAOM2B) encoding modified sarcosine oxidasewhere valine at position 94 was substituted with glycine in the aminoacid sequence of SEQ ID NO:1 was obtained by using pSAOEP3 and asynthetic oligonucleotide in SEQ ID NO:12 and a syntheticoligonucleotide complementary thereto, using QuickChange™ Site-DirectedMutagenesis Kit (supplied from Stratagene), and manipulating in the sameway as in the above.

A recombinant plasmid (pSAOM3B) encoding modified sarcosine oxidasewhere lysine at position 322 was substituted with arginine in the aminoacid sequence of SEQ ID NO:1 has was by using pSAOEP3 and a syntheticoligonucleotide in SEQ ID NO:13 and a synthetic oligonucleotidecomplementary thereto, and manipulating in the same way as in the above.

A recombinant plasmid (pSAOM4B) encoding modified sarcosine oxidasewhere valine at position 94 was substituted with glycine and glutamicacid at position 250 was substituted with glutamine in the amino acidsequence of SEQ ID NO:1 was obtained by using pSAOM2B and a syntheticoligonucleotide in SEQ ID NO:10 and a synthetic oligonucleotidecomplementary thereto, and manipulating in the same way as in the above.

A recombinant plasmid (pSAOM5B) encoding modified sarcosine oxidasewhere lysine at position 89 was substituted with arginine, valine atposition 94 was substituted with glycine and glutamic acid at position250 was substituted with glutamine in the amino acid sequence of SEQ IDNO:1 was obtained by using pSAOM4B and the synthetic oligonucleotide inSEQ ID NO:3 and the synthetic oligonucleotide complementary thereto, andmanipulating in the same way as in the above.

A recombinant plasmid (pSAOM6B) encoding modified sarcosine oxidasewhere lysine at position 89 was substituted with arginine, valine atposition 94 was substituted with glycine, serine at position 213 wassubstituted with proline and glutamic acid at position 250 wassubstituted with glutamine in the amino acid sequence of SEQ ID NO:1 wasobtained by using pSAOM5B and a synthetic oligonucleotide in SEQ ID NO:7and a synthetic oligonucleotide complementary thereto, and manipulatingin the same way as in the above.

A recombinant plasmid (pSAOM7B) encoding modified sarcosine oxidasewhere lysine at position 89 was substituted with arginine, valine atposition 94 was substituted with glycine, methionine at position 204 wassubstituted with alanine, serine at position 213 was substituted withproline and glutamic acid at position 250 was substituted with glutaminein the amino acid sequence of SEQ ID NO:1 was obtained by using pSAOM6Band a synthetic oligonucleotide in SEQ ID NO:14 and a syntheticoligonucleotide complementary thereto, and manipulating in the same wayas in the above.

A recombinant plasmid (pSAOM8B) encoding modified sarcosine oxidasewhere lysine at position 89 was substituted with arginine, valine atposition 94 was substituted with glycine, asparagine at position 166 wassubstituted with lysine, methionine at position 204 was substituted withalanine, serine at position 213 was substituted with proline andglutamic acid at position 250 was substituted with glutamine in theamino acid sequence of SEQ ID NO:1 was obtained by using pSAOM7B and asynthetic oligonucleotide in SEQ ID NO:5 and a synthetic oligonucleotidecomplementary thereto, and manipulating in the same way as in the above.

A recombinant plasmid (pSAOM9B) encoding modified sarcosine oxidasewhere lysine at position 89 was substituted with arginine, valine atposition 94 was substituted with glycine, asparagine at position 166 wassubstituted with lysine, methionine at position 204 was substituted withalanine, serine at position 213 was substituted with proline, glutamicacid at position 250 was substituted with glutamine, and lysine atposition 322 was substituted with arginine in the amino acid sequence ofSEQ ID NO:1 was obtained by using pSAOM8B and a syntheticoligonucleotide in SEQ ID NO:13 and a synthetic oligonucleotidecomplementary thereto, and manipulating in the same way as in the above.

EXAMPLE 3B Preparation of Modified Sarcosine Oxidases

Competent cells of Escherichia coli JM109 were transformed with eachrecombinant plasmid of pSAOM1B, pSAOM2B, pSAOM3B, pSAOM4B, pSAOM5B,pSAOM6B, pSAOM7B, pSAOM8B, and pSAOM9B to obtain the transformants.

Terrific broth (400 mL) was dispensed in a 2 L Sakaguchi flask,autoclaved at 121° C. for 20 min, cooled, and subsequently ampicillinseparately sterilized and filtrated was added at 100 μg/mL. A culturefluid (5 mL) of Escherichia coli JM109 (pSAOM1) previously cultured inLB medium containing 100 μg/mL of ampicillin at 30° C. for 16 hours wasinoculated to this medium, which was then cultured with aeration andstirring at 30° C. for 20 hours. At the completion of the culture, asarcosine oxidase activity was about 9.5 U/mL per 1 mL of the culturefluid in the above activity measurement.

The above microbial cells were collected by centrifugation, suspended in20 mM phosphate buffer (pH 7.5), subsequently disrupted ultrasonically,and further centrifuged to yield a supernatant as a crud enzymesolution. Nucleic acids were removed using polyethyleneimine from theresulting crude enzyme solution, ammonium sulfate fractionation wasgiven thereto, and then separation and purification was performed bydialyzing with 20 mM phosphate buffer (pH 7.5), applying on DEAESepharose CL-6B (supplied from Amersham Bioscience) and further treatingwith heat for one hour to yield a purified enzyme preparation. Thepreparation obtained by the present method exhibited a nearly singleband on SDS-PAGE. This mutant was designated as SAOM1B.

For transformants of Escherichia coli JM109 transformed with eachrecombinant plasmid of pSAOM2B, pSAOM3B, pSAOM4B, pSAOM5B, pSAOM6B,pSAOM7B, pSAOM8B, and pSAOM9B, a purified enzyme preparation wasobtained by the same way in the above, and the obtained enzymepreparation was each designated as SAOM2B, SAOM3B, SAOM4B, SAOM5B,SAOM6B, SAOM7B, SAOM8B, and SAOM9B.

EXAMPLE 4B Evaluation of Modified Sarcosine Oxidases

Various sarcosine oxidases obtained in Example 3B and ComparativeExample 1 were evaluated.

Action on proline was calculated from a relative ratio (%) of the enzymeactivity using L-proline as the substrate to the enzyme activity suingsarcosine as the substrate in the above activity measurement method. Kmvalues for sarcosine and L-proline were measured by changing thesubstrate concentration in the above activity measurement method. Theresults are shown in Table 5.

As is shown in Table 5, it has been confirmed that the reactivity toproline of the modified sarcosine oxidase of the present invention islowered compared with the unmodified one. The Km values of the modifiedsarcosine oxidase for sarcosine was nearly equal to or within 1.5 timesof the Km value of the unmodified sarcosine oxidase. Furthermore, it hasbeen shown that the modified sarcosine oxidase has at least either oneof the reactivity to proline of 0.7% or less or Km value of 150 mM ormore for L-proline as its property. TABLE 5 Acting Modified upon Kmvalue Km value one Mutant proline (proline) (sarcosine) SAOM1B K89R0.70% 151 mM 3.4 mM SAOM2B V94G 0.45% 214 mM 4.1 mM SAOM3B K322R 0.42%122 mM 4.7 mM SAOM4B V94G, E250Q 0.58% 213 mM 3.4 mM SAOM5B V94G, E250Q,K89R 0.55% 198 mM 3.3 mM SAOM6B K89R, V94G, S213P, 0.54% 210 mM 3.5 mME250Q SAOM7B K89R, V94G, 0.41% 203 mM 3.4 mM M204A, S213P, E250Q SAOM8BK89R, V94G, N166K, 0.41% 205 mM 3.4 mM M204A, S213P, E250Q SAOM9B K89R,V94G, N166K, 0.28% 202 mM 4.4 mM M204A, S213P, E250Q, K322R Unmodified —0.85% 142 mM 3.4 mM (control)

EXAMPLE 6B Effect of Proline on Reagent for Measuring Creatinine

Effects of proline when various sarcosine oxidases obtained in Example3B and Comparative Example 1 were applied to the reagent for measuringcreatinine were evaluated. To 300 μL of 50 mM PIPES-NaOH buffer (pH 7.5)containing 10 U/mL sarcosine oxidase (prepared in Example 3 andComparative Example 1), 1 mM dihydrogen disodium ethylenediaminetetraacetate, 50 mM sodium chloride, 0.1% (w/v) Triton X-100, 0.02%(w/v) 4-aminoantipyrine, 0.02% (w/v) TOOS (supplied from DojindoCorporate), 100 U/mL creatinine amidohydrolase (CNH-211, supplied fromToyobo Co., Ltd.), 50 U/mL creatine amidinohydrolase (CRH-221, suppliedfrom Toyobo Co., Ltd.), and 10 U/mL peroxidase (PEO-301, supplied fromToyobo Co., Ltd.), 10 μL of an aqueous solution of 5 mg/dL creatininewas added, reacted at 37° C., and changes of the absorbance at 546 nmwere measured using Hitachi 17060 type automatic analyzer. Using anaqueous solution of 100 mg/dL L-proline instead of the aqueous solutionof creatinine, the changes of the absorbance were measured by the sameway as in the above. The effect of proline was calculated by a relativeratio (%) of an absorbance increase for 5 min of the reaction usingL-proline as the substrate to an absorbance increase for 5 min of thereaction using creatinine as the substrate. The results are shown inTable 6. As is shown in Table 6, it has been confirmed that the effectof proline on the reagent is decreased by the use of the modifiedsarcosine oxidase of the present invention for the reagent for measuringcreatinine. TABLE 6 Modified Effect of one Mutant proline SAOM1B K89R5.7% SAOM2B V94G 3.6% SAOM3B K322R 3.5% SAOM4B V94G, E250Q 3.8% SAOM5BV94G, E250Q, K89R 3.5% SAOM6B K89R, V94G, S213P, E250Q 3.4% SAOM7B K89R,V94G, M204A, S213P, 3.2% E250Q SAOM8B K89R, V94G, N166K, M204A, 3.3%S213P, E250Q SAOM9B K89R, V94G, N166K, M204A, 1.9% S213P, E250Q, K322RUnmodified — 7.2% (control)

EXAMPLE 7B Effect of Proline on Reagent for Measuring Creatine

Effects of proline when various sarcosine oxidases obtained in Example3B and Comparative Example 1 were applied to the reagent for measuringcreatine were evaluated. To 300 μL of 50 mM PIPES-NaOH buffer (pH 7.5)containing 10 U/mL sarcosine oxidase (prepared in Example 3 andComparative Example 1), 1 mM dihydrogen disodium ethylenediaminetetraacetate, 50 mM sodium chloride, 0.1% (w/v) Triton X-100, 0.02%(w/v) 4-aminoantipyrine, 0.02% (w/v) TOOS (supplied from DojindoCorporate), 50 U/mL creatine amidinohydrolase (CRH-221, supplied fromToyobo Co., Ltd.), and 10 U/mL peroxidase (PEO-301, supplied from ToyoboCo., Ltd.), 10 μL of an aqueous solution of 5 mg/dL creatine was added,reacted at 37° C., and changes of the absorbance at 546 nm were measuredusing Hitachi 17060 type automatic analyzer. Using an aqueous solutionof 100 mg/dL L-proline instead of the aqueous solution of creatine, thechanges of the absorbance were measured by the same way as in the above.The effect of proline was calculated by a relative ratio (%) of anabsorbance increase for 5 min of the reaction using L-proline as thesubstrate to an absorbance increase for 5 min of the reaction usingcreatine as the substrate. The results are shown in Table 7. As is shownin Table 7, it has been confirmed that the effect of proline on thereagent is decreased by the use of the modified sarcosine oxidase of thepresent invention for the reagent for measuring creatine. TABLE 7Modified Effect of one Mutant proline SAOM1B K89R 5.3% SAOM2B V94G 3.1%SAOM3B K322R 3.2% SAOM4B V94G, E250Q 3.0% SAOM5B V94G, E250Q, K89R 3.4%SAOM6B K89R, V94G, S213P, E250Q 3.1% SAOM7B K89R, V94G, M204A, S213P,2.9% E250Q SAOM8B K89R, V94G, N166K, M204A, 3.1% S213P, E250Q SAOM9BK89R, V94G, N166K, 204A, 1.9% S213P, E250Q, K322R Unmodified — 7.0%(control)

1. Modified sarcosine oxidase which is a protein converted by adding,deleting, inserting or substituting at least one amino acid in an aminoacid sequence which constitutes a protein having a sarcosine oxidaseactivity, characterized by having the sarcosine oxidase activity andhaving improved stability in a liquid compared one before conversion. 2.(canceled)
 3. The modified sarcosine oxidase according to claim 1wherein the protein having the sarcosine oxidase activity has homologyof at least 50% or more to an amino acid sequence of SEQ ID NO:1. 4.(canceled)
 5. The modified sarcosine oxidase according to claim 1wherein the protein having the sarcosine oxidase activity has the aminoacid sequence of SEQ ID NO:1.
 6. The modified sarcosine oxidaseaccording to claim 1 characterized in that at least one amino acid in aregion corresponding to positions 155 to 250 in the amino acid sequenceof SEQ ID NO:1 is substituted with other amino acid.
 7. The modifiedsarcosine oxidase according to claim 1 characterized in that at leastone amino acid in a region corresponding to positions 82 to 92 or 354 to366 in the amino acid sequence of SEQ ID NO:1 is substituted with otheramino acid.
 8. The modified sarcosine oxidase according to claim 1characterized in that at least one amino acid selected from the groupconsisting of a region corresponding to positions 89, 155, 166, 204,213, 233, 240, 250 and 364 in the amino acid sequence of SEQ ID NO:1 issubstituted with other amino acid.
 9. The modified sarcosine oxidaseaccording to claim 1 characterized in that lysine at position 89 issubstituted with arginine in the amino acid sequence of SEQ ID NO:1. 10.The modified sarcosine oxidase according to claim 1 characterized inthat cysteine at position 155 is substituted with isoleucine in theamino acid sequence of SEQ ID NO:1.
 11. The modified sarcosine oxidaseaccording to claim 1 characterized in that asparagine at position 166 issubstituted with lysine in the amino acid sequence of SEQ ID NO:1. 12.The modified sarcosine oxidase according to claim 1 characterized inthat methionine at position 204 is substituted with alanine in the aminoacid sequence of SEQ ID NO:1.
 13. The modified sarcosine oxidaseaccording to claim 1 characterized in that serine at position 213 issubstituted with proline in the amino acid sequence of SEQ ID NO:1. 14.The modified sarcosine oxidase according to claim 1 characterized inthat cysteine at position 233 is substituted with serine in the aminoacid sequence of SEQ ID NO:1.
 15. The modified sarcosine oxidaseaccording to claim 1 characterized in that asparagine at position 240 issubstituted with tyrosine in the amino acid sequence of SEQ ID NO:1. 16.The modified sarcosine oxidase according to claim 1 characterized inthat glutamic acid at position 250 is substituted with glutamine in theamino acid sequence of SEQ ID NO:1.
 17. The modified sarcosine oxidaseaccording to claim 1 characterized in that alanine at position 364 issubstituted with valine in the amino acid sequence of SEQ ID NO:1. 18.Modified sarcosine oxidase which is a protein converted by adding,deleting, inserting or substituting at least one amino acid in an aminoacid sequence which constitutes a protein having a sarcosine oxidaseactivity, characterized by having the sarcosine oxidase activity andhaving a lowered action on L-proline compared with one beforeconversion.
 19. (canceled)
 20. The modified sarcosine oxidase accordingto claim 18 wherein the protein having the sarcosine oxidase activityhas homology of at least 50% or more to an amino acid sequence of SEQ IDNO:1. 21-24. (canceled)
 25. The modified sarcosine oxidase according toclaim 18 characterized in that at least one amino acid selected from thegroup consisting of positions 89, 94, and 322 in the amino acid sequenceof SEQ ID NO:1 is substituted with other amino acid.
 26. (canceled) 27.The modified sarcosine oxidase according to claim 18 characterized inthat valine at position 94 is substituted with glycine in the amino acidsequence of SEQ ID NO:1.
 28. The modified sarcosine oxidase according toclaim 18 characterized in that lysine at position 322 is substitutedwith arginine in the amino acid sequence of SEQ ID NO:1. 29-30.(canceled)
 31. Sarcosine oxidase having at least one property of thefollowings under a measurement condition at 37° C. and pH 8.0: action onL-proline: 0.7% or less based on sarcosine, and Km value for L-proline:150 mM or more. 32-34. (canceled)
 35. A gene encoding the modifiedsarcosine oxidase according to claim
 1. 36-41. (canceled)
 42. A geneencoding the modified sarcosine oxidase according to claim 18.